This subproject is one of many research subprojects utilizing the resources provided by a Center grant funded by NIH/NCRR. The subproject and investigator (PI) may have received primary funding from another NIH source, and thus could be represented in other CRISP entries. The institution listed is for the Center, which is not necessarily the institution for the investigator. In all eukaryotic cells, 26S proteasome catalyzes most intracellular protein degradation in an ATP dependent manner. The proteasome is composed of a 20S protease core particle (CP) sandwiched between two 19S regulatory complexes (RP). The proteasomal ATPases recognize the substrates targeted for the degradations, unfold globular substrates, induce gate-opening in the 20S, and facilitate translocation of the unfolded substrate into 20S CP for degradation. We are using single particle cryoEM together with other biochemical methods to study the mechanism of gate-opening in the 20S CP induced by the ATPases.